1. Field of the Invention
The invention pertains to recombinant Plasmodium expressing the SUB2 subtilisin-like protease which has a modified non-coding 3′UTR (3 prime untranslated region). The invention also related to methods for identifying virulent strains, and/or evaluating the virulence, of Plasmodium, and for screening new anti-malaria drugs are disclosed.
2. Description of the Related Art
Plasmodium is the parasite that causes malaria. The host-parasite relationship between malaria and its host involves a complex equilibrium between parasite multiplication, which is essential for its dissemination, and host responses1. Collateral damage attributable to the host-parasite interaction is responsible for the disease burden, morbidity and mortality in endemic areas where malaria is a major public health problem2.
The virulence of malaria is associated with the multiplication rate of the parasite and the ability of Plasmodium blood states to cytoadhere microvessels in deep organs1,3,4. While virulence factors involved in cytoadherence have been identified4, those involved in high multiplication rate, for which erythrocyte invasion by merozoite is a crucial step, remain poorly understood1,3,4.
Proteases are known to play a crucial role in various infectious diseases and cancer and have been successfully defined as chemotherapeutic targets. The specificity of protease activity permits the design of highly specific inhibitors which block a pathogenic process without considerable toxic side effects. Thus protease inhibitors are selectively toxic agents applicable for therapeutic use.
Biochemical and genetic analysis have shown that proteases and particularly serine proteases play a central role for the liberation and entry of merozoites into the host red blood cell.
Compared to intra-erythrocytic parasite states, the merozoite, as the sporozoite, is briefly free in the host plasma and therefore accessible to external factors including host immune factors like antibodies or to therapeutic drugs. The Merozoite Surface Protein 1 (MSP1) of Plasmodium is considered a promising candidate for a malaria vaccine. MSP1 undergoes a two step maturation process. The final processing of MSP1-42 occurs while the merozoite enters into the host RBC and is achieved by a parasite membrane-bound and calcium-dependent serine protease. Despite MSP1 polymorphism, this sequential processing is precisely conserved, even amongst different Plasmodium species. Even if the biological function linked to this maturation is still unknown, its inhibition using specific monoclonal antibodies or serine protease inhibitors efficiently blocks the RBC invasion.
It has recently been shown that the same parasite serine-protease is responsible for the second maturation step of the vaccine candidate Apical Membrane Antigen-1 (AMA1). Originally, AMA1 was identified to be the target of monoclonal antibodies which prevent RBC invasion by merozoites and more recently hepatocyte invasion by sporozoites. The inhibition of the maturation of AMA1 results in abortive RBC or hepatocyte invasion by the merozoites and the sporozoites, respectively.
Over the last decade, the establishment of Plasmodium genetic tools7, the genome sequencing of the laboratory clone P. falciparum 3D78 and various parasite transcriptome and proteome global projects9,10, have made profound changes in the field of malaria research. Interestingly, global expression profiling shows that 50% of the 5400 parasite genes are stage regulated, while the proportion of proteins predicted to be involved in gene regulation is low, especially when compared to S. cerevisiae8. Only a handful of regulatory elements have been identified11,12 and none is known that controls the multigene family coding for proteins mediating cytoadherence, which are subjected to antigenic variation.
A regulatory role for the respective introns of some malaria genes and 3′UTR (3 prime untranslated region) has been recently proposed13, as previously shown for the 3′UTR of the ookinete Pgs28 gene14. The 3′UTR, and particularly the addition of a polyadenylated tail, have been widely shown to play a central role in the regulation of eukaryotic15 and prokaryotic16 gene expression via the modulation of messenger RNA (mRNA) stability and translation initiation.
In malaria parasites, poly-A addition does not necessarily occur at the eukaryotic canonical AAUAAA (SEQ ID NO: 1) site and poly-A− mRNA have been identified17,18. In the absence of an inducible system to genetically study essential malaria genes, the deletion of 3′UTR was used to decrease gene expression, revealing interpretable phenotypes19,20. To evaluate in vivo the contribution of the crucial merozoite subtilisin-like P. berghei-SUB2 protease5,6 to the parasite life cycle, the inventors aimed to modulate Pbsub2 expression via the modification of its 3′UTR.